<Emphasis Type="Italic">In Vitro</Emphasis> susceptibility to selected antibiotics in bacteria of the <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> group

Susceptibility of strains of Bacteroides fragilis group (1284 isolates from cancerous and noncancerous patients in 1994–2004) showed an increase in resistance toward some antibiotics (by 9 % toward penicillin and 8 % toward clindamycin) compared with the resistance level of 10 years ago. The increase in resistance was not detected in the case of ampicillin + sulbactam and metronidazole.     

Effect of hyperbaric air on endotoxin from <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> strains

The aim of the project was to determine any effect of hyperbaric air on Bacteroides fragilis strains cultivated under hyperbaric conditions. Previously, it was hypothesized that there was a correlation between the presence of Bacteroides bacteria in patients preferring a meaty diet and cancer of the small intestine, and particularly of the large intestine and rectum. With respect to the fact that Bacteroides fragilis (BAFR) group are important producers of endotoxins, measurement and statistical evaluation of endotoxin production by individual strains of isolated Bacteroides species were used to compare bacteria isolated from various clinical samples from patients with colon and rectum cancer in comparison with strains isolated from other non-cancer diagnoses. Endotoxin production was proven by quantitative detection using the limulus amebocyte lysate (LAL) test in EU/mL. Production of endotoxins in these bacteria cultured under hyperbaric air conditions was higher than those strains cultured under normobaric anaerobic conditions. But these differences in endotoxin production were not statistically significant (t test with log-transformed data, p value = 0.0910). Based on a two-tier t test for lognormal data, it is possible to cautiously conclude that a statistically significant difference was found between endotoxin production by Bacteroides fragilis strains isolated from non-carcinoma diagnoses (strains (1–6) and strains isolated from colorectal carcinoma diagnoses (strains 7–8; Wilcoxon non-parametric test p = 0.0132; t test = 0.1110; t test with log-transformed data, p value = 0.0294).     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Betulin inhibits cariogenic properties of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by targeting <Emphasis Type="Italic">vicRK</Emphasis> and <Emphasis Type="Italic">gtf</Emphasis> genes

Streptococcus mutans, a multivirulent pathogen is considered the primary etiological agent in dental caries. Development of antibiotic resistance in the pathogen has created a need for novel antagonistic agents which can control the virulence of the organism and reduce resistance development. The present study demonstrates the in vitro anti-virulence potential of betulin (lup-20(29)-ene-3β,28-diol), an abundantly available plant triterpenoid against S. mutans UA159. Betulin exhibited significant dose dependent antibiofilm activity without affecting bacterial viability. At 240 µg/ml (biofilm inhibitory concentration), betulin inhibited biofilm formation and adherence to smooth glass surfaces by 93 and 71 % respectively. It reduced water insoluble glucan synthesis by 89 %, in conjunction with down regulation of gtfBC genes. Microscopic analysis confirmed the disruption in biofilm architecture and decreased exopolysaccharide production. Acidogenicity and aciduricity, key virulence factors responsible for carious lesions, were also notably affected. The induced auto-aggregation of cells upon treatment could be due to the down regulation of vicK. Results of gene expression analysis demonstrated significant down-regulation of virulence genes upon betulin treatment. Furthermore, the nontoxic effect of betulin on peripheral blood mononuclear cells even after 72 h treatment makes it a strong candidate for assessing its suitability to be used as a therapeutic agent.     

<Emphasis Type="Italic">egl</Emphasis>-<Emphasis Type="Italic">4</Emphasis> modulates electroconvulsive seizure duration in <Emphasis Type="Italic">C. elegans</Emphasis>

Increased neuronal excitability causes seizures with debilitating symptoms. Effective and noninvasive treatments are limited for easing symptoms, partially due to the complexity of the disorder and lack of knowledge of specific molecular faults. An unexplored, novel target for seizure therapeutics is the cGMP/protein kinase G (PKG) pathway, which targets downstream K⁺ channels, a mechanism similar to Retigabine, a recently FDA-approved antiepileptic drug. Our results demonstrate that increased PKG activity decreased seizure duration in C. elegans utilizing a recently developed electroconvulsive seizure assay. While the fly is a well-established seizure model, C. elegans are an ideal yet unexploited model which easily uptakes drugs and can be utilized for high-throughput screens. In this study, we show that treating the worms with either a potassium channel opener, Retigabine or published pharmaceuticals that increase PKG activity, significantly reduces seizure recovery times. Our results suggest that PKG signaling modulates downstream K⁺ channel conductance to control seizure recovery time in C. elegans. Hence, we provide powerful evidence, suggesting that pharmacological manipulation of the PKG signaling cascade may control seizure duration across phyla.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Dynamics of <Emphasis Type="Italic">Vulmar/VulMITE</Emphasis> group of transposable elements in <Emphasis Type="Italic">Chenopodiaceae</Emphasis> subfamily <Emphasis Type="Italic">Betoideae</Emphasis>

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia mangium</Emphasis>

A protocol was developed for Agrobacterium-mediated genetic transformation of Acacia mangium using rejuvenated shoots as the explant. Axillary buds and shoot apices of adult trees were rejuvenated by culturing them on Murashige and Skoog (MS) medium, and stem segments of rejuvenated shoots were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI121. The selection for transgenic shoots was performed through five consecutive steps on MS medium supplemented with 1.0 mg/l thidiazuron, 0.25 mg/l indole-3-acetic acid and different concentrations of geneticin (G418; 12–30 mg/l) and timentin (T; 50–300 mg/l) in the following order: 12 mg/l G418 and 300 mg/l T for 30 days, 20 mg/l G418 and 200 mg/l T for 60 days, 30 mg/l G418 and 100 mg/l T for 30 days, 12 mg/l G418 and 50 mg/l T for 30 days, and finally 15 mg/l G418 and 5 mg/l gibberellic acid (GA₃) for 60 days. Thirty-four percent of the stem segments produced resistant multiple adventitious shoot buds, of which 30% expressed the β-glucuronidase gene. The shoot buds were subjected to repeated selection on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine, 2.5 mg/l GA₃ and 20 mg/l G418. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 2.0 mg/l α-naphthaleneacetic acid, 0.1 mg/l kinetin and 20 mg/l G418. Genomic Southern blot hybridization confirmed the incorporation of the NPTII gene into the host genome.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.